Filter Paper Bridges Research Articles

The Effect of Media Types and NAA Concentrations on Agarwood (Aquilaria malaccensis Lamk.) Shoot Development in In Vitro Culture

<p>Agarwood (Aquilaria malaccensis Lamk.) is a forest product commodity with high economic value, but its availability in nature is decreasing due to deforestation and overexploitation. The availability of high-quality seedlings is necessary to meet the demand and conserve the sources of agarwood plants. Therefore, this study investigates the interaction effect between media types and 1-Naphthaleneacetic acid (NAA) concentrations on agarwood shoot development. It also aims to determine the best medium type and NAA concentration to stimulate agarwood shoot development. This research had two stages, namely the shoot induction and development. The shoot induction stage was conducted experimentally, using a completely randomized design (CRD) on a factorial treatment pattern of 2 factors i.e., the types of media and NAA concentrations. The shoots produced from the induction stage were cultured on Murashige-Skoog (MS) media with the same media types but without any NAA addition. Furthermore, the variables observed in both stages were the development of agarwood shoots, and the parameters measured included the number of shoots, number of leaves and shoot lengths. The results showed that the development of agarwood shoots was controlled by the type of medium used. Liquid MS medium supported with filter paper bridge produced the highest average number of shoots with 4.93 and 5.87 shoots explants-1 and length at 3.25 and 3.64 cm explant-1 in the induction and development stage, respectively. These findings will facilitate mass propagation of agarwood shoots, and in turn the availability of agarwood plantlets supports its conservation and production.</p>

In vitro seed germination and seedling performance of Hibiscus coddii subsp. barnardii

Abstract Hibiscus coddii subsp. barnardii is an endemic South African plant species with ornamental potential. The limited plant availability in nature and restrictions on the collection of living plant material (cuttings) for cultivation purposes, necessitated the development of a protocol for in vitro seed propagation to ensure all year round ex vitro establishment of plants. The effect of culture medium on seed germination and seedling performance was therefore studied. Various types of sterile seed cultures, namely moist filter paper bridges, five different strengths (¼, ½, –, full, 1¼) of PGR-free Murashige and Skoog medium (MS) and solid Gelrite® medium (control) were investigated. High germination percentages (85%-98%) were found in all three seed cultures, although seeds germinated on filter paper, Gelrite ® and lower strengths (¼, ½, –) of MS medium had shorter mean germination times (1.6-2.8 days) than those germinated on full and 1¼ strengths (3.5-4 days) of MS medium. Furthermore, the growth performance of seedlings varied with the strength of MS medium and was best supported on full strength MS medium, whereas ¼ and ½ strength MS media and Gelrite® cultures were detrimental for seedling growth.

Generation of Virus Free Potato Plantlets through Meristem Culture and Their Field Evaluation

Different aspects of micropropagation through meristem culture for the production of virus indexed source plants, in vitro tuberization and field evaluation of the in vitro regenerated plants were studied on four commercial cultivars of potato (Solanum tuberosum L.) viz., Diamant, Cardinal, Shilbilati and Lalpakri. The investigation was conducted at Rajshahi, Bangladesh from December 2010 to March 2012 to produce virus-free potato plantlets through meristem culture, shoot multiplications with root induction as well as their acclimatization and evaluation of morphological characters and tuber yield under field condition. Shoot tips of 25 - 30 day old field-grown plants of above mentioned four cultivars were used for meristem isolation. After isolation, meristems of these varieties of potato were cultured on “M” shaped filter paper bridge in Murashige and Skoog (MS) liquid medium. Four different treatments of media formulations viz. 0.1 mg/L KIN + 0.1 mg/L GA3, 0.1 mg/L KIN + 0.5 mg/L GA3, 0.5 mg/L KIN + 0.1 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used as plant growth regulators. From these formulations MS + 0.1 mg/L KIN + 0.5 mg/L GA3 was found to be the best for the primary establishment of meristem culture. The primarily established meristems were subcultured on to MS semisolid basal medium supplemented with four different treatment combinations of hormones viz. 0.5 mg/L BA + 1.0 mg/L IBA; 0.1 mg/L KIN + 0.1 mg/L GA3; 0.5 mg/L BA + 0.5 mg/L GA3 and 0.5 mg/L KIN + 0.5 mg/L GA3 were used to identify the suitable media compositions for shoot proliferation. Results showed that out of these four media treatments the formulation 0.5 mg/L BA + 0.5 mg/L GA3 was found to be the best suitable for shoot generation. Among the four cultivars of potato higher frequency of shoot proliferation (number of shoots/explant and longest shoot length) was observed in Diamant, though the highest shoot formation (76%) was recorded in Cardinal. Virus free in vitro grown potato plantlets were obtained through DAS-ELISA test and used substantially for micro-propagation. After gradual acclimatization of rooted plantlets of four potato cultivars, they were transferred into the field for cultivation and established successfully. It was observed from the field study of in vitro meristem-derived plantlets that there were no virus-affected plants. The virus-free exotic varieties were much superior in all vegetative attributes and yield compared to those of indigenous varieties with producing potato plants of normal height. In contrast, the indigenous varieties took a longer time to tuber initiation and maturity, lower plant height and number of leaves per plant, a higher number of tubers but a lower amount of tuber weight per plant, and poorer tuber grade than the exotic varieties. However, the variety Cardinal exposed the best performances in the context of survival percentage of plantlets (90%), days to tuber initiation (DTI), a number of leaves per plant (NL), tuber weight per plant (343.40%) and the percentage of rich tuber grade.

Effect of cold stratification and gibberellic acid on in vitro germination ofActinidia argutaandActinidia chinensis

The effect of cold stratification (CS) and gibberellic acid (GA3) alone and in combination on in vitro germination of Actinidia arguta and Actinidia chinensis seeds was studied. Sterile seeds of the two plant species were stratified at 4°C for 28 and 42 days. Batches of stratified and non-stratified (control) seeds from both plant species were germinated on plant growth regulator-free Murashige and Skoog (MS) media and also on sterile filter paper bridges moistened with dH2O and GA3 concentrations of 500, 1000, 1500, 2000 and 2500 ppm. In the two plant species, seeds from the control and the CS treatments alone did not germinate on MS medium. However on filter paper bridges, seeds cold stratified for 28 days yielded only 8 and 20% germination percentage (GP) in A. arguta and A. chinensis, respectively, whereas CS for 42 days did not promote germination. In A. arguta, treatments of non-stratified seeds with GA3 resulted in 91% optimum GP at 1572 ppm GA3 and optimum 29 days mean germination time (MGT) at 1678 ppm GA3. In A. chinensis, 48% optimum GP and 24 days optimum MGT were achieved at 2225 and 2033 ppm GA3 concentrations, respectively. Cold stratification (28 days) followed by GA3 treatments of A. arguta resulted in optimum GP of 88% and 23 days optimum MGT at GA3 concentrations of 1435 and 1610 ppm, respectively. The same combined treatment of A. chinensis seeds yielded optimum GP of 80% and optimum 25 days MGT at GA3 concentrations of 1565 and 2050 ppm, respectively. In contrast, CS (42 days) followed by GA3 in A. arguta resulted in optimum GP of 99% and 27 days optimum MGT at GA3 concentrations of 1563 and 2120 ppm, respectively. Similar treatments in A. chinensis seeds yielded optimum GP of 45% and optimum 23 days MGT at GA3 of 998 and 1629 ppm, respectively. In conclusion, CS alone had little effect on germination, whereas CS (28 days) followed by GA3 significantly (p≤0.05) improved GP in both plant species. Treatments with GA3 alone and CS (42 days) followed by GA3 were more effective on germination of A. arguta than A. chinensis. Germinated seeds on moist filter paper can produce seedlings when sub-cultured on MS medium for seedling development.

Establishment of seed germination and micropropagation protocols for Senegalia nigrescens, a potential anti-virulent species

ABSTRACTThis study describes seed germination and micropropagation of Senegalia nigrescens, an economic, medicinal and nitrogen-fixing species of South Africa. Seeds of S. nigrescens were subjected to pre-sowing treatments including soaking in cold water for 24 h, sulphuric acid (H2SO4) or hydrochloric acid (HCl) for 4, 8 and 12 min and mechanically scarifying seeds before sowing on Petri plates with wet filter paper. Mechanically-scarified seeds were also sown aseptically on a filter paper bridge, plain agar, ½ or full strength Murashige and Skoog (MS) basal medium. Single nodal explants from MS-derived seedlings were grown on MS media supplemented with 0.0, 0.5, 1.0 and 2.0 mg L−1 of benzylaminopurine (BAP) or kinetin (KIN) and a combination of 0.5 mg L−1 of BAP and KIN to investigate shoot multiplication. No significant differences were observed in the number of shoots produced across all treatments. However, the treatment containing 1.0 mg L−1 KIN produced a significantly higher shoot length (14.17 ± 5.20 mm) than 0.5, 1.0 and 2.0 mg L−1 BAP (7.67 ± 3.87, 6.75 ± 2.93 and 8.70 ± 3.56 mm, respectively). The highest rooting rate (16.7%) was obtained on ¼ strength MS supplemented with either indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) at concentrations 1.0 and 0.5 mg L−1, respectively. Rooted plantlets were successfully acclimatised with a 66.7% survival rate. The findings from this study would be of great benefit for the commercial propagation of S. nigrescens.

Synergetic effect of TDZ and BA on minimizing the post-exposure effects on axillary shoot proliferation and assessment of genetic fidelity in Rauvolfia tetraphylla (L.)

The stimulatory effect of different concentrations of thidiazuron (TDZ) on in vitro bud induction and proliferation of a potent medicinal woody shrub R. tetraphylla were investigated. Multiple shoots were induced from nodal explants on Murashige and Skoog medium augmented with different concentrations (0.2–1.0 µM) of TDZ. Continuous exposure beyond 5 weeks of singly supplemented TDZ resulted in hyperhydricity, fasciation and distortion of cultures. To avoid these deleterious effects, benzyl adenine (BA) was supplemented to the media. It not only mitigated the negative effects of prolonged TDZ exposure, but also enhanced shoot multiplication rate. About 30 shoots per explant with an average shoot length of 5.8 cm were obtained after 20 weeks of culture on MS medium fortified with 0.4 µM TDZ and 2.0 µM BA, regeneration frequency was also enhanced by 24% on this media as compared to singly supplemented TDZ. Microshoots (~ 5 cm) were successfully rooted on filter paper bridge in MS liquid medium augmented with different concentrations of indole-3-butyric acid (IBA) after 4 weeks of incubation. Best rooting response (80%) was achieved on MS medium supplemented with 0.3 µM IBA with (9.3 ± 1.45) mean number of roots and (3.3 ± 0.33 cm) root length per shootlet. The in vitro raised healthy plantlets with well developed root and shoot were successfully hardened off and acclimatized in thermocol cups containing sterilized planting substrate, Soilrite® for 8 weeks under culture room conditions prior to field transfer and successfully established in earthen pots with 90% survival rate. The genetic fidelity of in vitro regenerated plantlets was evaluated using random amplified polymorphic DNA markers. No polymorphism was observed in banding pattern among micropropagated and the donor plant, thus, exhibiting their genetic uniformity and clonal fidelity.

Comparative study on meristem culture of three potato cultivars diamant, cardinal and granula and their shoot formation

The main aim of this study was to analyze the comparative study on meristem culture of three important potato cultivars, diamant, cardinal and granula and their shoot formation using meristem tips. Apical meristems were isolated from shoot tips of 25-35 days old field grown plants. After surface sterilization the meristems (0.3 mm) were isolated from the shoot tips. After isolation the apical meristems were placed quickly on “M” shaped filter paper bridge in culture tubes containing liquid MS medium supplemented with various concentrations and combinations of different plant growth regulators. Meristems showed their first growth response by increase in size and became greenish white in colour. They continued their growth and developed shoots with roots. In the present investigation, among the three cultivars of potato, cardinal were the best responsive cultivars for the resuming new growth of cultured meristems on MS medium supplemented with KIN (0.4 mg l-1) + GA3 (0.5 mg l-1). Cultivar diamant showed comparatively better growth response in the primary culture and granula showed better results in shoot length formation in MS0 medium. Cultivar cardinal proved to be best potato cultivar in case of meristem culture than diamant and granula cultivars.Asian J. Med. Biol. Res. December 2015, 1(3): 537-544

<i>In Vitro</i> Seed Germination and Embryo Culture in <i>Nothapodytes foetida</i> (Wight) Sleumer

In vitro seed germination and embryo culture have been achieved in Nothapodytes foetida, this plant is known for its rich source of anticancer drug i. e., Camptothecin. In present study both normal and decoated seeds were subjected to different treatments viz., H2O, GA3, H2O2, H2SO4, chlorine water and mechanical scarification, further these were germinated on water agar medium (WA), filter paper bridge (FB), half strength MS (HMS) and full strength MS (FMS) medium. The highest percentage (69%) of germination was achieved from decoated seeds treated with 10mg/L GA3 and germinated on Filter Paper Bridge. And for embryo culture mature embryos were inoculated on MS medium containing various combination and concentrations of cytokinins (BAP, Kn and TDZ) and auxin (IAA and NAA) for rapid conversion into a plantlet. Among the different combinations of growth regulators; highest frequency (100%) of plantlet conversion was obtained on MS medium containing Kn (1.0mg/L) and NAA (0.2mg/L).

<i>In Vitro</i> Seed Germination and Embryo Culture in <i>Nothapodytes foetida</i> (Wight) Sleumer

In vitro seed germination and embryo culture have been achieved in Nothapodytes foetida, this plant is known for its rich source of anticancer drug i. e., Camptothecin. In present study both normal and decoated seeds were subjected to different treatments viz., H2O, GA3, H2O2, H2SO4, chlorine water and mechanical scarification, further these were germinated on water agar medium (WA), filter paper bridge (FB), half strength MS (HMS) and full strength MS (FMS) medium. The highest percentage (69%) of germination was achieved from decoated seeds treated with 10mg/L GA3 and germinated on Filter Paper Bridge. And for embryo culture mature embryos were inoculated on MS medium containing various combination and concentrations of cytokinins (BAP, Kn and TDZ) and auxin (IAA and NAA) for rapid conversion into a plantlet. Among the different combinations of growth regulators; highest frequency (100%) of plantlet conversion was obtained on MS medium containing Kn (1.0mg/L) and NAA (0.2mg/L).

In vitro mass propagation of Murraya koenigii L

Present endeavor was carried out for large scale shoots production from shoot tip meristem of Murraya koenigii obtained from mature field-raised plants. Apical shoot buds were cultured onto the MS medium augmented with various concentrations of single cytokinin (TDZ, BA and Kn) or in various combinations with an auxin NAA. Maximum percentage response (79%) with highest number of shoots (30.6±0.11) and shoot length (4.16±0.20cm) per explant was obtained on MS medium supplemented with TDZ (0.5μM)+NAA (0.3μM) after 8 weeks of culture. Shootlets with 3–4 nodes were utilized for in vitro rooting by using filter paper bridge method, and best response (60%) with root number (3.34±0.10) and root length (2.50±0.05cm) was appraised on 1/2 strength liquid MS medium supplemented with 1.5μM IBA after 4 weeks of incubation. The well-developed micropropagated plantlets were acclimatized successfully in the culture room for 70 days initially in soilrite under 150 PPFD (4 weeks) followed by transfer onto the mixture of soilrite+garden soil under 300 PPFD (6 weeks). The estimation of photosynthetic pigments (chl a/b and carotenoid content) and photosynthetic rate was found significantly increased after 2 weeks of acclimatization. Similarly, antioxidative enzymes SOD, CAT, APX and GR also showed an increasing trend throughout the hardening period. While contrarily, MDA and H2O2 contents accumulation were found decreasing showing a significant decrease in the internal stress in the plants. Hence, the outcome favors the hypothesis that to minimize the effects of oxidative stress and its products (MDA, H2O2, etc.), plants have evolved a complex ROS scavenging antioxidative enzymatic system. After 70 days acclimatization in culture room, the plants were transplanted to the field in normal garden soil showed good survival rate (80%) without any morphological variations.

Tissue culture of horse-radish (Cochlearia armoracia L.) meristems: sterilization of buds and comparison of media

The attempt was made to cultivate horse-radish meristems taken from buds removed from roots. The lowest per cent of contamination was found after the buds had been soaiked in 80% ethanol for 6 minutes and then in 5%, 7.5% or 10% chloramine for 30, 30 or 15 minutes, respectively. Both agar media: Murashige-Skoog and Linsmaier-Skoog, were equally good, providing a moderate number of developing plants. The Linsmaier-Skoog medium was more satisfactory when solidified with agar; the results with liquid medium and filter-paper bridges were not as good.

Bromelain Enzyme from Pineapple: In Vitro Activity Study under Different Micropropagation Conditions

The aim of this work was to evaluate the activity of bromelain in pineapple plants (Ananas comosus var. Comosus), Pérola cultivar, produced in vitro in different culture conditions. This enzyme, besides its pharmacological effects, is also employed in food industries, such as breweries and meat processing. In this work, the enzymatic activity was evaluated in the tissues of leaves and stems of plants grown in culture medium without plant growth regulator. The most significant levels of bromelain were observed in leaf tissue after 4 months of culture in vitro in medium with a filter paper bridge, followed by medium gelled by the agar. The results of this study, regarding the different structures of the pineapple (leaves and stems) in vitro showed that the activity of bromelain varied depending on the culture conditions, the time and structure of which was quantified, ensuring a viable strategy in the production of seedlings with high levels of bromelain in subsequent phases of micropropagation.

Assessment of genetic fidelity in Rauvolfia serpentina plantlets grown from synthetic (encapsulated) seeds following in vitro storage at 4 °C.

An efficient method was developed for plant regeneration and establishment from alginate encapsulated synthetic seeds of Rauvolfia serpentina. Synthetic seeds were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Llyod and McCown woody plant medium (WPM) and 100 mM calcium chloride. Re-growth ability of encapsulated nodal segments was evaluated after storage at 4 °C for 0, 1, 2, 4, 6 and 8 weeks and compared with non-encapsulated buds. Effects of different media viz; Murashige and Skoog medium; Lloyd and McCown woody Plant medium, Gamborg’s B5 medium and Schenk and Hildebrandt medium was also investigated for conversion into plantlets. The maximum frequency of conversion into plantlets from encapsulated nodal segments stored at 4 °C for 4 weeks was achieved on woody plant medium supplement with 5.0 μM BA and 1.0 μM NAA. Rooting in plantlets was achieved in half-strength Murashige and Skoog liquid medium containing 0.5 μM indole-3-acetic acid (IAA) on filter paper bridges. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. All the RAPD and ISSR profiles from generated plantlets were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. This synseed protocol could be useful for establishing a particular system for conservation, short-term storage and production of genetically identical and stable plants before it is released for commercial purposes.

High frequency shoot regeneration through cotyledonary node explants of Bauhinia tomentosa L., a woody leguminous tree

SummaryAn efficient in vitro method has been established for the production of whole plantlets of Bauhinia tomentosa using cotyledonary node (CN) explants excised from 15 d-old aseptic seedlings. Proliferating shoot cultures were obtained by placing CN explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), kinetin (Kn), and 2-isopentenyl adenine (2-iP), singly or in combination, with either of the auxins, indole-3-acetic acid (IAA) or αnaphthalene acetic acid (NAA). MS medium augmented with 5.0 µM BA was found to be most effective in providing the highest frequency (84%) of shoot regeneration, associated with the maximum number of shoots (9.0 ± 0.6) per explant. The highest efficiency of shoot proliferation was observed using a combination of 5.0 µM BA and 0.5 µM NAA, where the number of shoots increased to 16.2 ± 0.2 per explant after 8 weeks of culture. Improved shoot multiplication and elongation rates were achieved when 20.0 mg l–1 adenine sulphate (AdS) was added to the combined treatment of 5.0 µM BA and 0.5 µM NAA, where an average shoot number of 17.0 ± 0.6 and an average shoot length of 5.8 ± 0.3 cm were obtained.The maximum frequency of root formation (70%) and greatest root lengths (2.3 ± 0.4 cm) were obtained on filter-paper bridges in liquid MS medium augmented with 2.5 µM IBA and 5.0 µM chlorogenic acid (a phenolic compound) after 4 weeks of culture. Regenerated plantlets were successfully acclimatised in a growth room and subsequently transferred to a greenhouse.This is the first report on in vitro plantlet regeneration in B. tomentosa using CN explants.

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Petiole Explants of Cassia angustifolia Vahl.—a Potential Medicinal Plant

An effective protocol was developed for in vitro regeneration of the Cassia angustifolia via indirect organogenesis from petiole explants excised from 21-day-old axenic seedlings. Organogenic callus were induced on Murashige and Skoog (MS) medium supplemented with 5.0µM 2,4-dichlorophenoxy acetic acid and 2.5µM thidiazuron (TDZ). Adventitious shoot regeneration was achieved on MS medium supplemented with 5.0µM TDZ as it induced 8.5 ± 0.98 shoots in 85% cultures. The number of shoots and shoot length was significantly enhanced when cultures were subcultured on auxin-cytokinin-containing medium. The highest number of shoots (12.5 ± 1.10) and shoot length (4.3 ± 0.20cm) was recorded on MS medium supplemented with 5.0µM TDZ and 1.5µM indole-3-acetic acid. Regenerated shoots were rooted best on MS medium supplemented with 10.0µM indole-3-butyric acid followed by their transfer to liquid MS filter paper bridge medium. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 70% survival rate. The plants showed normal morphological characteristics similar to the field grown plants.

High efficiency in vitro plant regeneration from epicotyl explants of Ponkan Mandarin (Citrus reticulata Blanco)

Ponkan mandarin (Citrus reticulata Blanco) is one of the most important commercial cultivars of mandarin orange in China. This study reports an improved and efficient protocol for in vitro plant regeneration of Ponkan mandarin. Epicotyl segments, which were cut longitudinally into two halves, were used as explants. The shoot regeneration frequency was significantly increased by longitudinal cutting. A 100% shoot regeneration frequency and 13.2 shoots per explant were obtained when cultures were maintained in darkness for 20 d before being transferred to light conditions, with bud induction by indirect organogenesis. A 72.5% shoot regeneration frequency and 7.8 shoots per explant were obtained when explants were incubated under a 16-h light photoperiod continuously with buds differentiating directly from the cutting wound surface. The optimal medium for shoot formation was Murashige and Tucker basal medium supplemented with 2 mgL−1 BA and 30 gL−1 sucrose both under light conditions. The addition of the auxin NAA reduced the frequency of regeneration. A “filter-paper bridge” technique was used for rooting in this study. The basal portion of regenerated shoots was dipped into 1,000 mgL−1 IBA solution for 15 min before placement on a filter-paper bridge that was maintained in 1/2 MS liquid medium supplemented with 10 gL−1 sucrose. Eighty percent of the shoots rooted, and an average of 2.0 roots per shoot were achieved. Survival rate through acclimatization was 100%.

Micropropagation of Withania coagulans (Stocks) Dunal: A Critically Endangered Medicinal Herb

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l−1), Kn (0.5 mg l−1) and PG (0.5 mg l−1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l−1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l−1) and PG (0.5 mg l−1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l−1) + PAA (0.5 mg l−1) + CC (2 mg l−1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each.

Cultivo inicial in vitro de gemas axilares de Ananas comosus (L.) Merr., em meio líquido/sólido, na presença/ausência de luz

A capacidade regenerativa de gemas axilares de abacaxizeiro cv. Pérola (Ananas comosus (L.) Merr.) foi avaliada em meio de cultivo líquido e gelificado, na presença e ausência de luz, com o objetivo de produzir material para subsequentes fases da micropropagação. Os explantes foram estabelecidos, por 60 dias, em meio MS acrescido de 0,63 μM de ANA e 2,5 μM de BAP. O delineamento experimental foi inteiramente casualizado, constituído com quatro tratamentos (meio líquido com e sem ponte de papel de filtro e meio gelificado com ágar a 6,0 g L-1 e com Phytagel® a 3,0 g L-1), com quatro repetições e cinco explantes por repetição. Diferentes volumes de meio líquido (2,5 mL; 5,0 mL e 10,0 mL) também foram avaliados, usando-se quatro repetições por volume de meio de cultura. Maior número de brotos foi obtido na presença de luz, em meio líquido sem ponte de papel de filtro, onde 80% dos explantes se diferenciaram em brotos. Na ausência de luz, 68,8% a 87,5% dos explantes não regeneraram brotos. Explantes cultivados em volumes de 5,0 mL ou 2,5 mL de meio MS líquido, acrescido de 0,63 μM de ANA e 2,5 μM de BAP, na presença de luz, constitui um protocolo adequado para cultivo inicial de gemas axilares e regeneração de brotos de abacaxizeiro.

고추냉이 정단배양에 있어서 BA 농도 및 배양방법에 따른 기내증식 효과

고추냉이 (Wasabia japonica) 종자로부터 기내 무균상태 하에서 발아된 유묘에서 절취한 shoot-tip을 이용하여 대량증식 방법을 구명하기 위해 MS 기본배지에 BA 농도 및 반고체배지, filter paper bridge, 액체진탕배양 등의 배양방법을 달리하여 multi-shoot을 유도하였다. 1.0 mg/L BA가 들어있는 액체진탕배양에서 2주 동안 배양한 후 반고체배지에서 4주 동안 배양한 방법이 신초 수가 22.8개 그리고 신초의 길이가 3.5cm로 가장 양호한 결과를 보였으며 한 개의 shoot-tip으로부터 약 <TEX>$5{\sim}11$</TEX>개의 분할묘를 얻을 수 있었다. 또한 부정근을 유도하고자 여러 가지 농도의 IBA 및 NAA가 함유된 반고체배지에서 9주간 배양한 결과 배양 4주 이후부터 부정근이 출현하기 시작하였다. IBA (0.01 mg/L)가 NAA처리 보다는 부정근 형성에 효과가 있었으나 위의 두 생장조절물질이 전혀 첨가되지 않은 배지가 90.0%의 부정근 형성율을 보이면서 가장 양호한 것으로 나타났다. 잎과 뿌리가 잘 발달된 재분화묘를 원예용상토 (bio-media Co., peatmoss <TEX>$8{\sim}10%$</TEX>, coir dust <TEX>$66{\sim}70%$</TEX>, zeolite <TEX>$13{\sim}17%$</TEX>, vermiculite <TEX>$3{\sim}7%$</TEX>, perlite <TEX>$2{\sim}4%$</TEX>)에 넣어 2주간 습도를 높게 유지시킨 후 육묘온실에서 순화시킨 결과 모든 개체가 정상적인 형태로 성장하였다. Effect of BA concentrations and culture methods on in vitro plant multiplication from shoot-tip cultures of Wasabia japonica was studied. Shoot-tips with leaf primordia and apical meristem were cultured on MS basal medium for all the experiments. Liquid medium for 2 weeks followed by semi-solid medium for 4 weeks containing 1.0 mg/L BA was the best to number of shoots (22.8) and shoot length (3.5 cm). Shoots proliferated could be divided into ca. 5 to 11 of cultures for the multiplication of plantlets. Divided plantlets showed root formation (90%) well onto MS basal medium without growth regulators like IBA and NAA. After rooting, all the plantlets transferred into the pots containing composed soil (bio-media Co., peatmoss <TEX>$8{\sim}10%$</TEX>, coir dust <TEX>$66{\sim}70%$</TEX>, zeolite <TEX>$13{\sim}17%$</TEX>, vermiculite <TEX>$3{\sim}7%$</TEX>, perlite <TEX>$2{\sim}4%$</TEX>) and grown well into whole plants with multiple shoots.

The Influences of Growth Regulators and Culture Medium Composition on Shoot-Tip Cultures of Edible Canna

Explants obtained from the terminal buds of edible canna (Canna edulis Ker-Gawl.) were aseptically cultured with different combinations of auxins (NAA, IBA), cytokinins (BA, KN, 2ip), and culture media (B5, 1/2MS, MS), to establish a tissue culture system for this species. The combination of NAA or IBA with BA induced optimal explant growth, and when the explants grew substantially, TIBA promoted lateral shoot growth. For explant growth, the optimal concentration of NAA was 0.1∼0.5 mg L-1, irrespective of the BA concentration adopted, and the optimal concentration of BA was 0.5∼1 mg L-1. Some of the explants formed callus-like or protocorm-like tissues. Among the three culture media, B5 gave the highest survival rate of explants (92%), followed by 1/2 MS (72%) and MS (52%). In these combinations, micronutrients followed by macronutrients and vitamins, were found to be most important components for explant survival. Gellan gum at 2.5 g L-1 was a superior supporting/gelling material for explant growth compared to a filter paper-bridge with liquid medium or 4 g L-1 agar. Our results indicate that multiple plantlet propagation can be performed routinely, while callus formation remains a time-consuming endeavor.